Lens β-crystallins: the role of deamidation and related modifications in aging and cataract

Abstract

Crystallins are the major proteins in the lens of the eye and function to maintain transparency of the lens. Of the human crystallins, α, β, and γ, the β-crystallins remain the most elusive in their structural significance due to their greater number of subunits and possible oligomer formations. The β-crystallins are also heavily modified during aging. This review focuses on the functional significance of deamidation and the related modifications of racemization and isomerization, the major modifications in β-crystallins of the aged human lens. Elucidating the role of these modifications in cataract formation has been slow, because they are analytically among the most difficult post-translational modifications to study. Recent results suggest that many amides deamidate to similar extent in normal aged and cataractous lenses, while others may undergo greater deamidation in cataract. Mimicking deamidation at critical structural regions induces structural changes that disrupt the stability of the β-crystallins and lead to their aggregation in vitro. Deamidations at the surface disrupt interactions with other crystallins. Additionally, the α-crystallin chaperone is unable to completely prevent deamidated β-crystallins from insolubilization. Therefore, deamidation of β-crystallins may enhance their precipitation and light scattering in vivo contributing to cataract formation. Future experiments are needed to quantify differences in deamidation rates at all Asn and Gln residues within crystallins from aged and cataractous lenses, as well as racemization and isomerization which potentially perturb protein structure greater than deamidation alone. Quantitative data is greatly needed to investigate the importance of these major age-related modifications in cataract formation.

Keywords: Aging; Beta-crystallins; Cataracts; Deamidation; Post-translational modification; Proteomics.

Fig. 1

A. Reactions of the labile Asn/Asp residues in proteins from aged and cataractous lenses. Step A, formation of a succinimide ring structure and loss of ammonia. Step B, hydrolysis of succinimide ring and formation of L-Asp. Note: L-asp residues can also undergo the reverse reaction to form the succinimide ring. Step C, alternate hydrolysis of succinimide ring to form L-isoAsp. Step D, racemization of L-succinimide ring to the D-succinimide ring form. Steps E and F, hydrolysis of D- succinimde ring to either D-isoAsp or D-Asp, respectively. B. Backbone cleavage at Asn/Asp (shown for Asn). The cleavage reaction is in competition with formation of the succinimide ring shown in Fig. 1A.

Fig. 2

Calculation of percent single and double deamidation in peptide ITIYD-QENFQGK (αA-crystallin 33–44) from the water-insoluble fraction of a 93-year-old human lens by mixture model fitting. (A) The combined ion trace for the non-deamidated, singly-deamidated, and doubly deamidated forms of the peptide exhibiting a complex chromatogram due to deamidation, racemization, and isomerization. (B) MS scans from 34 to 42 min were averaged (experimental) and overlaid with fits to the calculated isotopic distributions for the unmodified, singly deamidated, and doubly deamidated peptide forms with their corresponding relative areas shown. (C) The composite fit resulting from the mixture modeling is shown overlaid with the experimental averaged mass spectrum to illustrate the quality of the fit (used with permission from Dasari et al., 2009).

Fig. 3

Schematic models of (A) βB2 homodimer (PBD:1YTQ), (B) truncated (tr) βB1 homodimer (PBD:1OKI), and (C) CPK model of βB2 homodimer (PBD:1YTQ). Dark blue, polypeptide chain A; light blue, polypeptide chain B; N- and C-td, N- and C-terminal domains.

Fig. 4

β-crystallin structures highlighting labile Asn/Gln that are deamidated in vivo. Ribbon model of (A) βB2 homodimer (PBD: 1YTQ) showing Gln70, Gln162, and Gln184. The light and dark blue color scheme represents the two homologous monomers. Ribbon model of (B) truncated (tr) βB1 homodimer (PBD:1OKI) showing Gln146, Gln157, and Gln204. Ribbon model of (C) βA4 monomer (PBD:3WLK) with residues homologous to Gln42, Asn54, Gln85, Asn120, Asn133, Asn155 and Gln180 found in βA3 highlighted. Dark blue, polypeptide chain A; light blue, polypeptide chain B; N- and C-td, N- and C-terminal domains. Deamidated residues shown in yellow. The numbering system is based on the amino acid sequence with the N-terminal methionine retained in βA3, but post-translationally removed in βB1 and βB2.

Fig. 5

Thermal denaturation at 55 °C of WTand Q204E βB1 adapted with permission from Lampi et al., 2002a, . (A) WT βB1 (blue curve) and Q204E βB1 (red curve) were heated with an equal molar amount of αA (dark blue and dark red, respectively). Additionally, Q204E βB1 was heated with αA in a molar ratio of 1:2 (brown).

Fig. 6

Conformational changes in WT βB2 due to deamidation at Gln70 adapted with permission from Takata et al., 2010. Differences in relative deuterium levels between WT βB2 and Q70E βB2 mutant peptides were overlaid onto the human βB2 crystal structure (PDB:1YTQ). The color of the peptides indicates the differential trend between the mutant and WT. Arrows indicate Gln70 shown in cpk model that was mutated to mimic deamidation. Gln70 in the N-td is oriented towards the C-td, disrupting the C-td and the interface. N- and C-td, N- and C-terminal domain.

Fig. 7

Proposed model for cataract formation adapted with permission from Lampi et al., 2012. During heat denaturation, both the N-terminal domain and C-terminal domain of βB2 partially unfold. The αA-chaperone forms a complex with the partially unfolded βB2, rescuing βB2 from precipitation. In contrast, deamidated βB2 precipitates under the same conditions due to a faster conversion to the more severely unfolded state and is unable to be rescued by α-crystallin.